Phenobarbital and methylcholanthrene stimulation of rat liver chromatin template activity

Vol. 33, No. 4, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PHENOBARBITAL AND METHYLCHOLANTHRENE STIMULATION OF RAT LIVER CRROMATIN TEMPLATE ACTIVITY Walter N. Piper and William P. Bousquet Department of Pharmacology and Toxicology Purdue University, Lafayette, Indiana 47907

Received

October

Both liver

10, 1968

phenobarbital

microsomal

This

drug

increased

indicated

have

shown that

after

than

chromatin

report

differences from

is

that

for

a more

activity

synthesis, Gelboin

after

template

from

rat

abolished

(1967) of

for

--in vitro

presented liver

or 3-methylcholanthrene

are

and

administration

The results

when compared

as

and Bresnick

effective

isolated

of

by Conney (1967).

(1960),

Madix

animals.

chromatin

RNA synthesis

in template

and Hultin

at 7 and 16 hours

of phenobarbital

template

as discussed

(1963).

from control

indicate

administration

effective

isolated

3-methylcholanthrene

RNA synthesis

the activity

is due to --de novo protein

of Von der Decken

chromatin

increase

enzymes,

and Conney and Gilman

the carcinogen

in

12 hours

is a more

to controls,

when basic

and that

proteins

are

removed

the chrome t in preparations.

Methods

:

Male

Holtzman

rats

i.p.

in saline)

(100 mg/kg; oil)

studies

(1961),

the present

metabolizing

enzyme activity

by the

Sokoloff

and 3-methylcholanthrene

3 and were

Livers ice.

were

perfused

to insure Basic of Marushige

by cervical

--in situ

removal proteins

given

saline

dislocation

with

ice-cold

from

liver

was isolated

Chromatin

were

(0.9%;

or 3-methylcholanthrene

sacrificed

Chromatin

(1966).

(150-200g)

was washed

3 times

(30 mg/kg; 12 hours

saline,

0.01 M tris,

phenobarbital i.p.

after

removed,

by the procedure with

i.p.),

in corn

injection.

and frozen

of Maruehige pH 8, prior

on dry and Banner

to dialysis

of sucrose. were

and Bonner

removed (1966).

from

chromatin

Samples

602

by a procedure

of chromatin

containing

simflar

to

that

5 mg of DNA

BIOCHEMICAL

Vol. 33, No. 4,1968

were

treated

Acid

insoluble

16,000

50 ml of ice-cold

material

x gavl

tris,

for

a Teflon

Aggregates

were

was aspirated,

referred

similar

moles

MnCli,

(specific

mixture

lysodeikticus

by addition

activity

1961),

and counted

15 minutes.

x M tris,

The superna-

and subsequently

in an RNA polymerase

tris

buffer,

system

pH 7.5,

CTP, GTP, and UTP,2

2.5 units

RNA polymerase

Elkhart,

were

incubated

Indiana)

at 30°C for

10% TCA.

5% TCA, and dissolved

was added

at 78,000

(1964).

each of ATP.

of 2 ml of ice-cold

aliquot

in 25 ml of 0.01

1.25 p

0.4 pc C14-ATP,

from Micrococcus , and chromatin

as DNA

of 0.5 ml.

mixtures

2 ml of ice-cold

at

in 6 ml of 0.01

content,

of 50 p moles

Laboratories,

in a volume

Reaction

et al.,

20 mC/mM),

(Miles

was resuspended

template

consisted

0.2 p moles activity

template

for

of Nakamato,

The assay

suspended

and was centrifuged

DNA and protein

bath.

of the mixture

at 2200 x gav for

for

in an ice

chromatin.

was assayed

to that

pellet

centrifuged

to as acid-treated

Chromatin

was then

hand homogenizer,

assayed

20 minutes

by centrifugation

Chromatin

The gelatinous

2 hours.

pH 8.

0.2 N HCl for

was collected

20 minutes.

pH 8, with

J3av for

tant

with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Precipitates

radioactivity

were

and were washed

in 1.0 ml of 0.1 N NaOH.

to 15 ml XDC scintillation for

10 minutes,

solution

in a Beckman

(Bruno

LS-100

stopped

twice

with

An 0.8 ml

and Christian,

liquid

scintillation

spectrometer. DNA was determined method

of Lowry,

s

a.,

by the method

of Diache

(1955),

and protein

by the

(1951).

Results: Both template 1

phenobarbital activity

Spinco

for

and 3-methylcholanthrene RNA synthesis,

as shown

treatment in Figure

increase

the

1.

#30 rotor.

n

‘ The following abbreviations were used: AMP, adenosine-5’-monophosphate; ATP, CTP, GTP, DTP, the three 5’ triphosphotes of adenosine, cytosine, guanosine and uridine; TCA, trichloroacetic acid.

603

M

Vol. 33, No. 4, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

604

BIOCHEMICAL

Vol. 33, No, 4, 1968

Differences removed,

phenobarbital

ability

that

activity

as ,shown in Figure

Both its

in template

protein

is

Experiments alteration

as a template involved

Toxicology

for

being

protein

was

the National

Institute

thus

indicate

activity.

to determine

Project

increasing

The data

of template

by phenobarbital

by Program

chromatin,

RNA synthesis.

conducted

produced

was supported

from

when basic

modify

in the differences

are currently

work

abolished

2.

of the template

This

were

and 3-methylchokanthrene

to function

basic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the nature

of the

and 3-methylcholanthrene. Grant

of General

No.

Medical

15,005

in Pharmacology-

Sciences.

References: Bruno,

G.A.,

and Christian,

J.E.,

Conney,

A.H.,

Pharmac.

Conney,

A.H.,

and Gilman,

Dische, Vol.

Z., In: The Nucleic 1, p. 285, Academic

Gelboin,

H.V.,

Rev.,

Madix,

J.C.,

Marushige, Nakamoto,

l9,

A.G.,

and Bresnick, K.,

T.,

Von der Decken,

and Bonner, Fox,

CF.,

J. Biol.

L.,

33,

1216 (1961).

Chem.,

Science, Farr,

E.,

Biochem.

J.,

J. Mol.

and Weiss, T.,

3,

3682 (1963).

ed. by E. Chargaff New York, 1955.

N.J.,

A. and Hultin,

Chem.,

317 (1967).

Acids, Press,

and Sokoloff,

Lowry, O.H., Rosenbrough, l93, 265 (1951).

Anal.

134,

A.L.,

and Randall,

Biol.,

Arch.

605

J.

Davidson,

611 (1961).

Biophys.

S.B.,

and J.N.

Res. l5,

Biol.

Biochem.

R.J.,

J. Biol,

Chem.,

Comm., 2S, 445 (1967).

160 (1966). Chem., Biophys.,

239,

167 (1964).

9G, 201 (1960).